To track protein expression, localization, or conformational changes as components of cellular signaling pathways, biologists need general tools for the in vivo site-specific labeling of proteins with fluorophores or other useful probes. Traditional chemical methods rely on the nucleophilicity of cysteine or lysine side chains and are too promiscuous for in vivo use, and genetic methods such as fusion to green fluorescent protein (GFP) carry bulky payloads (GFP is 238 amino acids) and are limited in the color range and nature of the spectroscopic readout.
A survey of the existing methods for targeting small molecules to protein sequences reveals that the shorter the target sequence, the less specific the conjugation chemistry. For instance, very specific conjugation can be achieved by fusing the protein O6-alkylguanine-DNA alkyltransferase (AGT) to the target protein of interest, and then adding a fluorescently-labeled O6-benzylguanine suicide substrate for the AGT. (Keppler, A. et al. Nat. Biotechnol. 21, 86–89, 2003). However, the AGT tag is 207 amino acids and introduces a large amount of steric bulk. Smaller peptide tags are more desirable, but difficult to target with small molecules with high specificity. For example, cysteine labeling is not at all specific inside cells, and tetracysteine labeling (Griffin, B A et al. Science 281, 269–272, 1998), while much better, is still insufficiently specific for most applications and allows only a small set of probes to be introduced. Transglutaminase is already used to label glutamine side chains with fluorophores in vitro (Sato, H. et al. Biochemistry 35, 13072–13080, 1996), however it is relatively promiscuous for peptide and protein substrates, precluding its use in mammalian cells. In vitro labeling and microinjection has the disadvantage that protein localization and abundance may be altered. Polyhistidine tag methodology has the disadvantage that nickel is toxic, promiscuous, membrane impermeant and a quencher of fluorescence.
Accordingly, there exists a need for a method to label proteins and peptides that is specific and which offers a variety of a labeling options.